Genomic analysis of Shigella isolates from Lebanon reveals marked genetic diversity and antimicrobial resistance

In this study, we characterized 54 clinical isolates of Shigella collected in North Lebanon between 2009 and 2017 through phenotypic and genomic analyses. The most prevalent serogroup was S. sonnei, accounting for 46.3 % (25/54) of the isolates, followed by S. flexneri (27.8 %, 15/54), S. boydii (18.5 %, 10/54) and S. dysenteriae (7.4 %, 4/54). Only three isolates were pan-susceptible, and 87 % (47/54) of the isolates had multidrug resistance phenotypes. Notably, 27.8 % (15/54) of the isolates were resistant to third-generation cephalosporins (3GCs) and 77.8 % (42/54) were resistant to nalidixic acid. 3GC resistance was mediated by the extended-spectrum beta-lactamase genes bla CTX-M-15 and bla CTX-M-3, which were present on various plasmids. Quinolone resistance was conferred by single point mutations in the gyrA DNA gyrase gene, leading to GyrA S83L, GyrA D87Y or GyrA S83A amino acid substitutions. This is the first study, to our knowledge, to provide genomic insights into the serotypes of Shigella circulating in Lebanon and the various antimicrobial resistance determinants carried by these strains.


INTRODUCTION
Shigella, a Gram-negative bacterium from the family Enterobacteriaceae, is a major cause of diarrhoeal disease, accounting for about 210 000 deaths annually [1,2].Shigella is mostly transmitted through contaminated food, water or person-to-person contact.It can cause mild diarrhoea, severe dysentery with bloody stools and potentially fatal dehydration, especially in vulnerable populations, such as young children, the elderly and immunocompromised individuals [3,4].
The genus Shigella encompasses four serogroups: S. dysenteriae, S. boydii, S. flexneri and S. sonnei.Cases of shigellosis in low-and middle-income countries (LMICs), where the disease burden is highest, are mostly caused by the last two of these serogroups [1,[4][5][6].Despite the public health impact of shigellosis, there is currently no licensed vaccine against Shigella, primarily due to its substantial genomic and phenotypic diversity, which poses a major challenge in vaccine development [7,8].Consequently, the management of shigellosis relies on supportive care and antimicrobial therapy.However, the increasing frequency of antimicrobial resistance (AMR) in Shigella strains has become a major concern, leading the World Health Organisation (WHO) to classify Shigella as a priority pathogen for which new antimicrobial drugs are urgently required [2,[9][10][11].
According to global disease burden estimates for 2016, the incidence of episodes of diarrhoea attributable to Shigella among children under the age of 5 years was 107.3 episodes per 1000 child-years in the Middle East and North Africa [1].In Lebanon, the national burden of Shigella infections remains unclear as the dysentery cases reported by the Ministry of Public Health (https:// www.moph.gov.lb/en/Pages/2/194/surveillance-data) group together the infections caused by Entamoeba histolytica and those caused by Shigella spp.This lack of clarity is compounded by the limited epidemiological data for Shigella in Lebanon due to the lack of robust and sustainable epidemiological surveillance programmes and the challenges faced by most clinical laboratories in the identification of Shigella and its differentiation from enteroinvasive Escherichia coli (EIEC) [12,13].A study recently conducted in two Lebanese tertiary healthcare settings showed that EIEC and Shigella accounted for 18.9 % of the enteric pathogens isolated from patients with acute community-acquired diarrhoea [12].Lebanon is currently grappling with calamitous challenges to its infrastructure and political stability, together with pollution, associated with a severe economic collapse that resulted in an acute devaluation of its currency by more than 95 % and shortages of imported goods, limiting the availability and increasing the prices of essential goods, with an impact on public health, including critically important antimicrobial drugs (CIAs) [14].Furthermore, the country is currently providing shelter for about 1.5 million refugees displaced by the ongoing conflict in Syria [15].In this context, an understanding of the distribution of Shigella serotypes and associated antimicrobial susceptibility patterns in Lebanon is crucial for epidemiological surveillance and vaccine development, which is also a WHO priority [7].We therefore performed phenotypic and in-depth genomic analyses of Shigella isolates collected in North Lebanon to provide baseline information on the distribution of Shigella serotypes and AMR determinants in the country.

Shigella isolates
Fifty-four Shigella spp.isolates were obtained from clinical stool samples of patients admitted to tertiary healthcare facilities for bacillary dysentery between 2009 and 2017 [Nini Hospital (n=37), Al-Haykal Hospital (n=3), Dar Al-Chifae Hospital (n=4), El Youssef Hospital Centre (n=4) and Tripoli Governmental Hospital (n=6)].The isolates from Nini Hospital identified as Shigella spp.were collected throughout the study period, whereas those from El Youssef Hospital were collected from April 2015.Isolates were collected on a random basis from the other hospitals.Isolates were initially identified with API 20E strips (bioMérieux)

Impact Statement
Shigellosis poses a significant threat to public health, particularly in low-and middle-income countries, where it predominantly affects young children.The severity of the illness, coupled with the ability of Shigella to acquire multiple antimicrobial resistance determinants, further exacerbates the problem.Effective surveillance of shigellosis is recognized as crucial for informed decision-making and appropriate public health interventions.However, the epidemiology of Shigella remains poorly understood in Lebanon, a country facing unique challenges of economic instability and poor healthcare infrastructure.We addressed this gap in our knowledge by characterizing 54 Shigella isolates from North Lebanon, focusing on their prevalence, antimicrobial susceptibility profiles and genotypic characteristics.Our findings reveal considerable diversity among the circulating Shigella strains, with a high proportion displaying antimicrobial drug resistance.Resistance to third-generation cephalosporins was mediated by bla CTX-M-15 and bla CTX-M-3 genes carried on IncI1 and IncFIB plasmids.Our findings highlight the critical need for ongoing surveillance efforts to provide an accurate assessment of the burden of disease due to shigellosis and to provide essential guidance for its management.The whole-genome sequencing data presented here will also serve as a valuable resource for future research investigating the evolutionary patterns of antimicrobial susceptibility in Lebanon, thereby deepening our understanding of the mechanisms and dynamics underlying the spread of Shigella strains in the region.
or with the RapID ONE system (Remel).They were then transported to the Laboratoire Microbiologie Santé et Environnement (LMSE) in Tripoli, Lebanon, where they were stored in the Collection Microbiologique de l'Université Libanaise (CMUL).For characterization and further analyses, the isolates were subsequently shipped to the French National Reference Centre for Escherichia coli, Shigella and Salmonella (FNRC-ESS) at the Institut Pasteur, Paris, France, in accordance with international regulations.
Five extended-spectrum beta-lactamase (ESBL)-producing Shigella isolates were selected and sequenced with a Nanopore MinION sequencer (Oxford Nanopore Technologies) as previously described [9].Briefly, DNA was extracted from the isolates grown overnight in alkaline nutrient agar at 37 °C, and then cultured in brain-heart infusion (BHI) broth at 37 °C with shaking to a final OD 600 of 0.8.The bacterial cells were then harvested, and genomic DNA was extracted with Qiagen Genomic-tip 100 G −1 columns, according to the manufacturer's protocol.The extracted DNA was used to prepare a library according to the instructions of the 'Native barcoding genomic DNA (with EXP-NBD104, EXP-NBD114, and SQK-LSK109)' procedure provided by Oxford Nanopore Technology.Sequencing was then performed with a MinION Mk1C device.Long reads were filtered with Filtlong (version 0.2.0), 95 % of the reads being retained on the basis of a minimum length of 1000 bp (https://github.com/rrwick/Filtlong).The genome sequences were assembled with UniCycler version 0.4.8 according to a hybrid approach using both the short and long reads [19].Prokka version 1.14.5 (https://github.com/tseemann/prokka)was used for plasmid annotation [20].Illumina short reads were mapped to constructed plasmids with BWA version 0.7.4 [21] and SAMtools version 1.13 [22].

Shigella serotype distribution
Shigella serotyping was performed by slide agglutination and in silico with ShigaPass.No discrepancies were observed between the results of the two methods.S. sonnei was the most prevalent serogroup ( All isolates could be classified to the previously identified Shigella cgMLST clusters [24].The S. sonnei isolates belonged to lineage 3, particularly to subclade 3.6.1 (80 %, 20/25), known as 'CipR parent' (Data S1) [11].Three of the six S. flexneri group 1-5, X, Y isolates belonged to phylogroup (PG) 3, the other three belonging to PG1a (Data S1) [2,24].These results highlight the diversity of the Shigella serotypes circulating in North Lebanon and demonstrate the predominance of S. sonnei.We also detected serotypes that had emerged more recently, such as S. boydii 20 and S. flexneri 1c. S. boydii 20 was first described in 1999, and it became the predominant serotype of S. boydii in Canada within a year [31].The emergence of S. flexneri 1c has also been reported in Bangladesh, Egypt, Indonesia and Pakistan [32][33][34].
Our study has several limitations.The number of Shigella isolates collected was limited because not all the participating tertiary healthcare centres sent all the Shigella isolates they obtained to the LMSE.In addition, shigellosis is generally a self-limiting disease with symptoms such as watery diarrhoea, and these characteristics may have resulted in many of those infected not seeking medical attention or undergoing testing.Moreover, many clinical laboratories experience difficulties identifying Shigella spp. with the available testing tools.Finally, this was not a nationwide study as all the participating laboratories were located in northern Lebanon.The sample studied may not therefore be entirely representative of the true prevalence and distribution of Shigella strains in the population.

Analysis of the AMR genes
In addition to performing antimicrobial susceptibility testing, we also analysed WGS data to investigate the mechanisms of resistance in more detail.Genomic analysis revealed the presence of 19 different AMR genes, including genes conferring resistance to sulfonamides (sul1 and sul2), tetracycline (tetA and tetB), trimethoprim (dfrA1, dfrA5 and dfrA14), and phenicols (catA1).Several different AMR genes were implicated in resistance to aminoglycosides.The most frequent were strA, strB and aadA1, found in 44.4 % (24/54), 44.4 % (24/54), and 42.6 % (23/54) of the isolates, respectively.Moreover, 46.3 % (25/54) of the isolates carried more than one aminoglycoside resistance gene (Table 1, Fig. 1, Data S1).Quinolone resistance was present at high frequency (77.8 %, 42/54) and was mediated by a single mutation in the quinolone resistance-determining region (QRDR) of the gyrA gene encoding subunit A of the DNA gyrase.The GyrA S83L variant was the most frequent, found in 64.3 % (27/42) of the nalixidic acid-resistant isolates (MIC >16 mg l −1 ), followed by the GyrA D87Y variant, which was present in 33.3 % (14/42).The GyrA S83A variant was found in only one isolate.All isolates carrying the gyrA S83L mutation (20 S. sonnei, six S. boydi 20 and one S. flexneri 2a) had decreased susceptibility to ciprofloxacin (MIC >0.06 mg l −1 and ≤0.5 mg l −1 ).By contrast, only 21.4 % (3/14) of the isolates carrying the gyrA D87Y mutation displayed decreased susceptibility to ciprofloxacin, all the remaining isolates carrying this mutation being susceptible (MIC ≤0.06 mg l −1 ).This mutation was found in all nine S. flexneri 6 isolates and in five S. sonnei isolates.The S. dysenteriae 3 isolate with the gyrA S83A mutation was also susceptible to ciprofloxacin.Only two isolates had plasmid-mediated quinolone resistance (PMQR) genes.These two isolates -one S. boydii 10 (CL-045) and one S. boydii 20 (CL-056) -harboured the qnrS1 gene.CL-056 also carried the gyrA S83L mutation.The combined presence of the qnrS1 gene and the gyrA mutation in CL-056 led to resistance to ciprofloxacin (Table 1, Fig. 1, Data S1).
Ciprofloxacin-resistant isolates were rare in our study (a single isolate), but 55.6 % (30/54) of the Shigella isolates displayed decreased susceptibility to this drug, corresponding to the first step towards the development of full resistance to ciprofloxacin through the accumulation of QRDR mutations and/or PMQR genes [39].In our study, 80 % (20/25) of the S. sonnei isolates -the most prevalent Shigella serogroup/serotype in our study -belonged to sublineage 3.6.1 (also known as 'CipR parent').This S. sonnei sublineage originated in South Asia in 2007 and has displayed a tendency to accumulate mutations in the QRDR region of the gyrA and parC genes, generating genotypes resistant to ciprofloxacin [9][10][11].Caution should therefore be exercised in the use of fluoroquinolones to treat shigellosis in Lebanon.
Ampicillin resistance was conferred by the bla OXA-1 and bla TEM-1B beta-lactamase genes, the latter of which predominated (60 %, 9/15).The bla OXA-1 gene was carried by six isolates (three S. flexneri and three S. dysenteriae), along with at least the tet(B), aadA1 and catA1 genes (Table 1, Fig. 1, Data S1).This combination is normally seen in the SRL pathogenicity island (PAI) conferring resistance to tetracycline, aminopenicillins, streptomycin and chloramphenicol [2,40].Resistance to 3GCs was conferred by two ESBL genes, bla CTX-M-15 and bla CTX-M-3 , which were found in 27.7 % (15/54) of Shigella isolates (eight S. sonnei, four S. flexneri and three S. boydii isolates).The bla CTX-M-15 gene was the most common, present in 10 of the 15 isolates, the CTX-M-3 gene being found in the remaining five isolates (Table 1, Fig. 1, Data S1).Previous studies have also reported a high prevalence of the bla CTX-M-15 gene in Shigella isolates, this gene being considered the most common ESBL gene in Shigella [41,42].It is usually found in association with the bla TEM-1B gene, and this was the case in all but two of the isolates studied here [33,[43][44][45][46][47][48].Matar et al. reported the first detection of CTX-M-15-producing Shigella in Lebanon in 2007 [49].The presence of both bla CTX-M-15 and bla TEM-1B has been documented not only in Shigella isolates, but also in ESBL-producing E. coli collected from river water and from wastewater from refugee camps in Lebanon [49][50][51][52].

Characterization of ESBL-encoding plasmids
We initially investigated the location of the ESBL genes in short-read assemblies, with PlasmidFinder, Resfinder and BLASTn, by determining whether these genes were present on the same contig as the plasmid replicons.Using this approach, we found that all ESBL genes were, indeed, present on the contig carrying the plasmid replicon genes, confirming that they were plasmid-borne.Two different Inc-type plasmids were associated with the ESBL genes: IncI1 (found in 11/15 isolates resistant to 3GCs) associated with bla CTX-M-15 or bla CTX-M-3 , and IncFIB (found in the remaining four isolates) associated with bla CTX-M-15 (Fig. 1, Data S1).One of the short-read assemblies (from S. boydii 10 isolate CL-045) revealed that the 111 026 bp contig carrying the IncFIB replicon was 99 % identical, over 100 % of the alignment, to p38 (GenBank accession no.CP099775.1),a plasmid from an S. sonnei isolate (S17BD05916) collected in Belgium in 2017 [53].This alignment confirmed the presence of a complete ~111 kb IncFIB plasmid harbouring the bla CTX-M-15 gene proximal to the IS1380 family transposase ISEcp1.We whether the other three isolates bla CTX-M-15 (CL-015, CL-056, CL-063) harboured the same IncFIB plasmid, by performing both read-mapping analysis (Table S1) and BLASTn analysis with BRIG on draft genomes against plasmid p38 (Fig. 2).There was a high degree of identity between these isolates, and all displayed >96.5 % coverage of the IncFIB plasmid, confirming the presence of very similar plasmids in each of these isolates (Fig. 2, Table S1, Data S1).This plasmid was carried by S. boydii isolates belonging to three different serotypes and by one S. flexneri 6 isolate (Figs 1 and 2, Data S1).
However, it proved challenging to obtain a complete sequence for the IncI1 plasmid.We addressed this problem by performing long-read sequencing on five isolates, to explore the genetic characteristics of this plasmid further.The read-mapping and BLASTn (using BRIG) analyses carried on the five circularized plasmids and the six draft assemblies of the 11 ESBL-producing isolates carrying an IncI1 plasmid revealed a high degree of identity and more than 97 % coverage of two different IncI1 plasmids (Figs 3  and 4, Table S1).The first, a plasmid of about 87 kb, carried the bla CTX-M-3 gene flanked by a remnant ISEcp1 and the IS6 family transposase IS26.(Table 2).This plasmid was carried by S. sonnei and S. flexneri (serotypes 2a and 6) isolated from two different hospitals between 2011 and 2016 (Figs 1 and 3, Data S1).It was highly similar (100 % identity, with 98 % coverage) to p7111-69 (GenBank accession no.CP049176), from an S. sonnei isolate (7111.69)acquired in Turkey in 2019 (Fig. 3) [43].The same plasmid  2).It was predominantly found in S. sonnei isolates but was also carried by one S. flexneri serotype 1c isolate (Figs 1 and 4, Data S1).This plasmid was 99 % identical, with >97 % coverage, to p4 (GenBank accession no.CP099782.1)from an S. sonnei isolate (S14BD05406) collected in Belgium in 2015 (Fig. 4) [53].
The presence of the same IncI1/IncFIB plasmids in different Shigella serogroups (S. sonnei, S. flexneri and S. boydii) isolated from different countries suggests that these plasmids have been successfully transmitted horizontally between different strains and even across continents.This transfer mechanism raises concerns about the dissemination of AMR genes, highlighting the urgent need for enhanced surveillance and the importance of prudent antimicrobial drug use to mitigate the dissemination of antimicrobial drug resistance determinants.To our knowledge, this is first to provide insight into the of Shigella circulating in Lebanon and the AMR determinants they carry.It revealed a high degree of genetic diversity in the circulating Shigella strains, with a marked prevalence of MDR isolates.Our findings also suggest possible interactions between ESBL-producing Shigella strains circulating globally and the strains present in Lebanon, although further studies are required to characterize this phenomenon in more detail.Future studies with a larger sample size and enhanced surveillance efforts are warranted to obtain a comprehensive understanding of Shigella epidemiology and AMR patterns in Lebanon and beyond.

Fig. 1 .
Fig. 1.Maximum-likelihood phylogeny of 54 Shigella genomic sequences isolated from Lebanon.The phylogenetic tree was reconstructed with the 'create SNP project' tool in EnteroBase, based on the 81 516 non-repetitive SNPs present in 95 % or more of all the queried genomes.The strips show the associated information for each isolate, in the following order from left to right: (1) serogroup identity with the serotype indicated inside; (2) source of the isolate; (3) year of isolation; (4) antimicrobial resistance genes; and (5) plasmid type associated with the ESBL genes.Bar, 0.001 nucleotide substitutions per site.

Table 1 .
Genomic characteristics and antibiotic resistance profiles of the Shigella isolates analysed in this study

Table 2 .
Complete plasmids obtained by long-read sequencing in our study